NK Cell Activation/

Expansion Kit
human
Order no. 130-094-483

Contents Loaded Anti-Biotin MACSiBead Particles are subsequently used

for the expansion of NK cells. Best activation and expansion of NK
1. Description cells is accomplished by using one loaded Anti-Biotin MACSiBead
1.1 Principle of the NK Cell Activation/Expansion Kit Particle per two cells (bead-to-cell ratio 1:2). The cells are cultured
1.2 Background information for further expansion.
1.3 Applications NK cells, activated by using Anti-Biotin MACSiBead Particles, can
1.4 Reagent and instrument requirements be used for any downstream processing such as cytokine analysis,
cytolytic activity, gene expression, or functional studies.
2. Protocol
2.1 Loading of Anti-Biotin MACSiBead Particles MACS Anti-Biotin MACSiBead Particles show no autofluorescence
and normally do not need to be removed prior to flow cytometric
2.2 Magnetic separation of NK cells using the NK Cell
analysis. However, if desired, removal of Anti-Biotin MACSiBead
Isolation Kit
Particles is easily achieved by using the MACSiMAG Separator
2.3 NK cell activation and expansion protocol (see 2.6).
2.4 Immunofluorescent staining
3. Examples of NK cell activation and expansion using the NK 1.3 Applications
Cell Activation/Expansion Kit Activation and expansion of resting NK cells from peripheral
blood mononuclear cells (PBMCs) or after isolation with the
1. Description NK Cell Isolation Kit (#130-092-657) or CD56 MicroBeads
(#130-050-401).
This product is for research use only.
Components 2 mL MACS Anti-Biotin MACSiBead 1.4 Reagent and instrument requirements
Particles, cell culture grade, corresponding
Buffer: Phosphate-buffered saline (PBS) pH 7.2, supplemented
to 410 MACSiBead Particles; MACSiBead
Particles conjugated to monoclonal anti-biotin with 0.5% human serum albumin (HSA) and 2mM EDTA.
antibodies. Keep buffer cold (28 C). Degas buffer before use, as air
bubbles could block the column.
0.4
mL CD335 (NKp46)-Biotin, human
Note: HSA can be replaced by other proteins such as bovine serum albumin,
functional grade (100g/mL). fetal calf serum or human AB serum. Buffers or media containing Ca 2+ or Mg2+
0.4
mL CD2-Biotin, human
functional are not recommended for use.
grade (100g/mL). NK Cell Isolation Kit (#130-092-657) or CD56 MicroBeads
Product format All components are supplied in azide-free buffer, (#130-050-401).
Anti-Biotin MACSiBead Particles contain MACS Columns and MACS Separators: Choose the
stabilizer. appropriate MACS Separator and MACS Columns according
Storage Store protected from light at 28C. Do not to the number of labeled cells and to the number of total cells.
freeze. The expiration date is indicated on the
Column Max. number Max. number Separator
vial label. of labeled cells of total cells

Positive selection
1.1 Principle of the NK Cell Activation/Expansion Kit
MS 10 210 MiniMACS, OctoMACS,
TThe NK Cell Activation/Expansion Kit is designed to VarioMACS, SuperMACSII
activate and expand human NK cells. The kit consists of LS 10 210 MidiMACS, QuadroMACS,
Anti-Biotin MACSiBead Particles and biotinylated antibodies VarioMACS, SuperMACSII
against human CD335 (NKp46) and CD2. Anti-Biotin MACSiBead
Particles loaded with biotinylated antibodies are used to activate Note: Column adapters are required to insert certain columns into the
and expand resting NK cells purified from human blood or PBMCs. VarioMACS or SuperMACS II Separators. For details refer to the respective
MACS Separator data sheet.
1.2 Background information
Medium: NK MACS Medium (# 130-107-879) supplemented
In a first step the Anti-Biotin MACSiBead Particles are loaded with with 5% AB serum and 500 IU/mL human Interleukin 2 (IL-2).
biotinylated antibodies. Best activation is achieved by using equal
Human IL-2, e.g., Human IL-2 IS, premium grade (#130-097-
amounts of the provided biotinylated antibodies against CD335
748).
(NKp46) and CD2.
Note: Other combinations of biotinylated antibodies may be (Optional) MACS GMP Cell Expansion Bags (#170-076-403)
experimentally tested for their suitability, if required. or flat bottom cell culture plates with lids.

Miltenyi Biotec GmbH Miltenyi Biotec Inc.

140-002-663.05

Friedrich-Ebert-Strae 68, 51429 Bergisch Gladbach, Germany 2303 Lindbergh Street, Auburn, CA 95602, USA
Phone +49 2204 8306-0, Fax +49 2204 85197 Phone 800 FOR MACS, +1 530 888 8871, Fax +1 877 591 1060
macs@miltenyibiotec.de macs@miltenyibiotec.com
www.miltenyibiotec.com
page 1/3

Humidified incubator.
MACSmix Tube Rotator (#130-090-753) for loading of
MACSiBead Particles.
(Optional) Fluorochrome-conjugated antibodies for flow
cytometric analysis. For more information about antibodies
refer to www.miltenyibiotec.com/antibodies.
(Optional) Propidium Iodide Solution (#130-093-233) or
7-AAD for flow cytometric exclusion of dead cells.
2. Protocol
All steps in the protocol have to be performed under sterile
conditions.
2.1 Loading of Anti-Biotin MACSiBead Particles
Resuspend Anti-Biotin MACSiBead Particles thoroughly by
vortexing before use, to obtain a homogenous suspension.
Anti-Biotin MACSiBead Particles are supplied without
preservative. Remove aliquots under aseptic conditions.
It is recommended to load Anti-Biotin MACSiBead Particles in
batches of 110 Anti-Biotin MACSiBead Particles. Loaded Anti-
Biotin MACSiBead Particles are stable for up to 2 months when
stored at 28 C.
1. Pipette 100 L of CD335 (NKp46)-Biotin and 100 L CD2-
Biotin into sealable 2 mL tube and mix well.
Note: This antibody combination is optimized for achieving maximum NK
cell activation and expansion.
2. Resuspend Anti-Biotin MACSiBead Particles thoroughly by
vortexing.
3. Remove 500 L Anti-Biotin MACSiBead Particles (110 Anti-
Biotin MACSiBead Particles) and add to antibody mix.
4. Add 300 L buffer to adjust to a total volume of 1 mL.
Note: Anti-Biotin MACSiBead Particles can be loaded in a flexible manner
with biotinylated antibodies or ligands other than those supplied. If desired,
add other biotinylated antibodies or ligands at appropriate concentrations and
adjust with buffer to a total volume of 1 mL, accordingly.
5. Incubate for 2 hours at 28 C under constant, gentle rotation
by using the MACSmix Tube Rotator at approximately 4 rpm
(slowest permanent run program).
6. The loaded Anti-Biotin MACSiBead Particles (110 Anti-
Biotin MACSiBead Particles/mL) are now ready to use. Do not
remove the loaded Anti-Biotin MACSiBead Particles from
the antibody mix. Store at 28 C for up to 2 months.
2.2 Magnetic separation of NK cells using the NK
Cell Isolation Kit
Isolate the NK cells according to the NK Cell Isolation Kit data
sheet.
2.3 NK cell activation and expansion protocol
This NK cell activation and expansion protocol is optimized for NK
cells that have been purified using the NK Cell Isolation Kit, using
one loaded Anti-Biotin MACSiBead Particle per two NK cells
(bead-to-cell ratio 1:2).
Note: Other ratios than 1:2 of loaded Anti-Biotin MACSiBead Particles per
cell may be required for other applications.
Unless otherwise specifically indicated, all Miltenyi Biotec products and services
140-002-663.05
are for research use only and not for diagnostic or therapeutic use.
Order no. 130-094-483
Volumes for activation given below are for 10 purified NK cells
or 10 PBMCs. When working with higher cell numbers, scale up
all reagent volumes and total volumes, accordingly (e.g. for 210
total cells, use twice the volume of all indicated reagent volumes
and total volumes).
1. Resuspend loaded Anti-Biotin MACSiBead Particles
thoroughly and transfer 5 L (510 loaded Anti-Biotin
MACSiBead Particles) per 10 NK cells to a suitable tube.
Note: : If unloaded MACSiBead Particles shall be used for negative control
experiments, replace loaded Anti-Biotin MACSiBead Particles by adding 510
of unloaded Anti-Biotin MACSiBead Particles per 106 NK cells.
2. Add 100 L culture medium to the loaded Anti-Biotin
MACSiBead Particles and centrifuge at 300g for 5 minutes.
3. Aspirate supernatant and resuspend loaded Anti-Biotin
MACSiBead Particles in 50 L of fresh culture medium.
4. Resuspend PBMCs or purified NK cells at a density of 106
cells per 950 L of culture medium (NK MACS Medium
supplemented with 5% AB serum and 500 IU/mL IL-2).
5. Add the prepared Anti-Biotin MACSiBead Particles from step
3 to the 950 L of cell suspension and mix well.
6. Add the mixture to a suitable cell culture vessel at a density
of 10 cells per mL, for example, in the wells of a 24 well cell
culture plate.
7. Incubate at 37 C and 5% CO.
Inspect cultures daily, and add fresh medium if required.
For NK cell expansion the addition of culture medium is
required regularly. In the following a guideline for the stimulation
and expansion of NK cells is described.
8. At day 6, gently pipette cell suspension up and down to break up
clumps.
9. Determine cell number and dilute to 11.510 cells per
mL by adding fresh culture medium (NK MACS Medium
supplemented with 5% AB serum and 500 IU/mL IL-2).
Transfer to a fresh culture vessel of appropriate size.
NK cell expansion is donor-dependent. Best results are achieved
when NK cells are maintained in a cell density of 11.510 NK cells
per mL. Depending on the expansion rate it might be necessary to
split culture daily.
2.4 Immunofluorescent staining
Volumes for fluorescent labeling given below are for 10 total
cells. When working with fewer than 106 cells and up to 10 cells,
use the same volumes as indicated.
MACSiBead Particles show no autofluorescence and do not need
to be removed prior to flow cytometric analysis.
Scatter properties of cells may be altered due to strong interaction
between cells and MACSiBead Particles.
1. Resuspend cells to break up cell clumps.
2. Wash cells by adding 12 mL of buffer per 10 cells and centrifuge
at 300g for 10 minutes. Aspirate supernatant completely.
3. Add 10 L of each staining antibody, e.g., CD3-FITC
(#130-080-401) and CD56-PE (#130-090-755) to 10 cells
resuspended with buffer to a total volume of 110 L.
page 2/3
 Order no. 130-094-483

4. Mix well and incubate for 10 minutes in the dark in the
refrigerator (28 C).
Note: Working on ice requires increased incubation time. Higher temperatures
and/or longer incubation times lead to non-specific cell labeling.
5. Wash cells by adding 12 mL of buffer per 10 cells and
centrifuge at 300g for 10 minutes. Aspirate supernatant
completely.
6. Resuspend cell pellet in a suitable amount of buffer for analysis
by flow cytometry or fluorescence microscopy.
B) NK cell expansion rates
Anti-Biotin MACSiBead Particles were loaded with CD335
(NKp46) and CD2 antibodies. NK cells were isolated using the
NK Cell Isolation Kit and expanded using 1 loaded Anti-Biotin
MACSiBead Particle per 2 NKcells. Cells were cultured in
NK MACS Medium supplemented with 5% AB serum and
500IU/mL IL-2 at an initial density of 10 NK cells per mL. Cells
were expanded for 18 days.
For comparison NK cells were cultured in medium supplemented
with 5% AB serum and 500 IU/mL IL-2 alone.

3. Example of NK cell activation and expansion

using the NK Cell Activation/Expansion Kit 100

A) Cytotoxicity of expanded NK cells 90

Anti-Biotin MACSiBead Particles were loaded with CD335 (NKp46) 80

and CD2 antibodies. NK cells were activated and expanded from

Cell expansion (n-fold)

70
PBMCs using one loaded MACSiBead particle per viable cell. Cells
were cultured in NK MACS Medium supplemented with 5% AB 60
serum and 500 IU/mL IL-2. After 2 weeks, NK cells were incubated 50
with K562 target cells in different ratios. In addition, NK cells
40
incubated in NK MACS Medium supplemented with 5% AB serum
and 500 IU/mL IL-2 alone were analyzed. Specific target cell lysis 30
by NK cells was determined after 4 hours.
20

10
100
0
6 8 10 12 14 16 18 20
90
Time (d)
80

70
Killed target cells (%)

Unstimulated Stimulated
60

50
Refer to www.miltenyibiotec.com for all data sheets and protocols.
40

30
Warranty
The products sold hereunder are warranted only to be free from defects in workmanship
20
and material at the time of delivery to the customer. Miltenyi Biotec GmbH
makes no warranty or representation, either expressed or implied, with respect to
10
the fitness of a product for a particular purpose. There are no warranties, expressed
or implied, which extend beyond the technical specifications of the products.
0
Miltenyi Biotec GmbHs liability is limited to either replacement of the products or
0 2 4 6 8 10 12
refund of the purchase price. Miltenyi Biotec GmbH is not liable for any property
Ratio E:T damage, personal injury or economic loss caused by the product.

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Unless otherwise specifically indicated, all Miltenyi Biotec products and services page 3/3
140-002-663.05

are for research use only and not for diagnostic or therapeutic use.